Recombinant human proteins corresponding to their natural amino acid sequences have been used for the treatment and diagnosis of a broad range of human diseases since the 1980s. However, most recombinant human proteins do not survive long enough in vivo and are rapidly cleared from circulation. For example, proteins with a molecular mass less than 20 kDa have been reported to be filtered at the level of renal tubules, often leading to a dose-dependent nephrotoxicity. The short in vivo half-life of these proteins compromises their natural biological functions, requiring higher doses or more frequent administration, which in turn impairs patient compliance and increases the burden on health care providers. These clinical demands merit the search and development of therapeutic proteins with longer circulation half-life.
In addition to the direct mutations of individual protein structure for achieving longer half-life (e.g. ARANESP™ by Amgen and TNKnase by Genentech), two systemic approaches have been used for the creation of therapeutic proteins with longer half-life. One is “PEGylation”, which refers to chemical cross-linking of polyethylene glycol (PEG) compounds to target proteins. PEG-bound proteins have larger molecular sizes and are more slowly cleared from the circulation. PEGylation has been clinically demonstrated and recognized by the biotech industry as a standard method of extending the half-life of various target proteins. A shortcoming of PEGylation is the significant impairment of the biological activity of target proteins. The altered structure of PEGylated proteins also risks generating an immunogenic response in the human body.
Another systemic approach is the genetic fusion of target therapeutic protein(s) with another human carrier protein to stabilize the target protein in circulation in the form of a fusion protein complex. Two ideal human carrier protein candidates for fusion with therapeutic proteins are human immunoglobulin and albumin. Both immunoglobulin and albumin are very stable and abundant in blood. Fusion proteins comprising a therapeutic protein and either immunoglobulin or albumin would theoretically retain the biological activity of the therapeutic protein, be more stable in circulation than the therapeutic protein alone, and be completely homologous to natural human proteins, minimizing the risk of immunogenic responses [1,2].
One practical strategy with this approach is to genetically fuse a therapeutic protein with an Fc fragment of a human immunoglobulin [1, 3, 4]. Modern bioengineering technology has successfully created fusion proteins consisting of a therapeutic protein, such as cytokines and soluble receptors, and an Fc fragment of immunoglobulin G (IgG) [5-26]. For example, IL-10, an anti-inflammatory and anti-rejection agent, has been fused to the N-terminal of murine Fc.gamma.2a to increase IL-10's short circulating half-life [9]. In another example, the N-terminal of human IL-2 has been fused to the Fc portion of human IgG 1 or IgG 3 to overcome the short half life of IL-2 and its systemic toxicity [26]. Two fusion proteins comprising an Fc fragment have been successfully developed as biomedicines and approved by FDA for the treatment of rheumatoid arthritis and chronic plaque psoriasis [27, 28, 29].
Human IgG is composed of four polypeptides (two identical copies of light chain and heavy chain) covalently linked by disulfide bonds. The proteolysis of IgG by papain generates two Fab fragments and one Fc fragment. The Fc fragment consists of two polypeptides linked by disulfide bonds. Each polypeptide, from the N-terminal to C-terminal, is composed of a hinge region, a CH2 domain and a CH3 domain. The structure of the Fc fragment is nearly identical across all subtypes of human immunoglobulin. IgG is one of the most abundant proteins in the human blood and makes up 70 to 75% of the total immunoglobulin in human serum. The half-life of IgG in circulation is the longest among all five types of immunoglobulin and may reach 21 days.
Disulfide bonds formed between thiol groups of cysteine residues play an important role in the folding and stability of proteins, usually when proteins are secreted to an extracellular medium. The disulfide bond stabilizes the folded form of a protein in several ways. First, it holds two portions of the protein together, biasing the protein towards the folded state. Second, the disulfide bond may form the nucleus of a hydrophobic core of the folded protein, i.e., local hydrophobic residues may condense around the disulfide bond and onto each other through hydrophobic interactions. Third, and related to the first and second points, by linking two segments of the protein chain and increasing the effective local concentration of protein residues, the effective local concentration of water molecules is lowered. Since water molecules attack amide-amide hydrogen bonds and break up secondary structures, disulfide bonds stabilize secondary structure in their vicinity. For example, researchers have identified several pairs of peptides that are unstructured in isolation, but adopt stable secondary and tertiary structure upon forming a disulfide bond between them. The native form of a protein is usually a single disulfide species, although some proteins may cycle between a few disulfide states as part of their function. In proteins with more than two cysteines, non-native disulfide species, which are almost always unfolded, may be formed.
A flexible junction region of the fusion protein which allows the two ends of the molecule to move independently plays a very important role in retaining each of the two moieties' functions separate and efficient. Therefore, the junction region should act as a linker which combines the two parts together, and as a spacer which allows each of the two parts to form its own biological structure and not interfere with the other part. Furthermore, in order to avoid the induction of immunogenicity, the junction region should be native to the human body and simple in structure [5, 25].
The primary structure of the hinge region of immunoglobulin includes three cysteines, such as cys223, cys229 and cys232 in the case of the human IgG 1 structure used by the present inventors. While the cys229 and cys232 form two interchain disulfide bonds by binding between counterparts of the two chains, the cys223 remains free. Therefore, it is highly possible that this free cysteine may bind with another intrachain or interchain cysteine, to form a non-native disulfide bond in the protein maturation process upon secretion from host cells or during subsequent purification. This non-native disulfide bond may not only alter the structure and conformation of the therapeutic protein, but may also interfere with the biological activity of the therapeutic protein or induce harmful immunogenicity when the fusion protein is administrated into the human body.
Many therapeutic proteins such as erythropoietin (EPO) and granulocyte macrophage colony-stimulating factor (GM-CSF) have a cysteine near their C-terminal. The role of this cysteine in maintaining proper structure and function has yet to be well-defined. The cysteine proximal to the C-terminal may be essential for maintaining proper structure, facilitating correct folding or retaining normal biological activity. The inventors hypothesize that if proteins with a cysteine near its C-terminal are fused to the natural sequence of the hinge region of a Fc fragment, the very limited space between the last cysteine of the C-terminal of the fused protein and the first cysteine of the N-terminal of the Fc fragment (cys223) may lead to the formation of an unexpected disulfide bond between these two cysteines. The formation of the unexpected disulfide bond may alter the structure and/or the folding of the fused protein component as well as alter the flexibility of the hinge region. As a result, normal functions of the fused therapeutic protein in the fusion protein complex may be impaired.
Even if the target therapeutic protein does not contain a cysteine near its C-terminal, another cysteine in its structure may, after three dimensional folding, become sufficiently close to the free cysteine (e.g. cys223) of the hinge region to form a non-natural disulfide bond that may alter the structure and biological activity of the fused target protein. The inventors' hypothesis may partially explain why there has yet to be any clinically-proven success in attempts to create functional fusion proteins with widely-used growth factors such as EPO, G-CSF and GM-CSF, etc.
Previous reports have used various methods to create fusion proteins between a therapeutic protein and an Fc fragment/immunoglobulin molecule. In most of these reports, researchers changed amino acid sequences of the target protein, added a linker peptide between the C-terminal of the target protein and the N-terminal of the hinge region of Fc fragment, or truncated the hinge region of the Fc fragment of the hinge region (resulting in the removal of the free cysteine (e.g. cys223)).
In U.S. Pat. No. 5,908,626, a fusion protein of IFN β with a human immunoglobulin Fc fragment is described which was linked by a synthetic oligopeptide (GGS)2(GGGS)2 [6]. The inventors in that patent believe this linker can “reduce the possibility of generating a new immunogenic epitope (a neoantigen) at what would otherwise be the fusion point of the IFN β and the immunoglobulin Fc fragment”. In U.S. Pat. Nos. 6,797,493, 6,900,292, 7,030,226, 7,226,759, and 7,232,668, the hinge region was replaced by a 16-amino acid peptide linker GS(GGGS)3GS [10, 12, 13, 20, 21]. In addition to the genetic approach, chemical manipulation has also been used to address the problem of non-native disulfide bonds. For example, the inventors in U.S. Pat. No. 6,808,902 developed a process for treating an IL-1ra-Fc fusion protein with a copper (II) halide in order to prevent or correct a non-native disulfide bond which caused misfolding of that fusion protein [12]. An Fc-EPO fusion protein (rather than the conventional EPO-Fc fusion) has shown poor pharmacokinetics and little EPO efficacy in mice; mutation of four amino acids of the EPO molecule is required to obtain a functional Fc-EPO fusion protein [30].
As mentioned above, the hinge region plays the role of the flexible junction region between the fused therapeutic protein and the Fc fragment (CH2 and CH3). Truncation or significant changes of the hinge region may have undesirable effects on ability of the hinge region to act as flexible junction. The addition of peptide linkers may not only impair the natural conformation of the fusion protein but also greatly increase the risk of immunogenicity by introducing a non-native structure.
The need exists for therapeutic protein/Fc fragment fusion proteins that have a prolonged half-life and/or enhanced activity without increasing the risk of an immunogenic response.